The impact of different preceding crops on soil nitrogen structure and nitrogen cycling in tobacco-planting soil

Soil nitrogen content, structure, and nitrogen cycling play a crucial role in tobacco growth quality, with different preceding crops having varying impacts on tobacco cultivation soil. This study conducted using field experiments, employed three treatments with different preceding crops, namely tobacco, barley, and rapeseed, to investigate the effects of different preceding crops on soil nitrogen structure and the expression levels of soil nitrogen cycling-related functional genes in tobacco cultivation soil. The results indicated that different preceding crops had varying effects on the content of different nitrogen forms in tobacco cultivation soil. Ammonium nitrogen and nitrate nitrogen were the two nitrogen forms which were most influenced by preceding crops, with the ammonium nitrogen content in soils following barley and rapeseed preceding crops increasing by 82.88% and 63.56%, respectively, compared to sole tobacco cultivation. The nitrate nitrogen content in tobacco cultivation soil was 26.97% higher following barley preceding crops and 24.39% higher following rapeseed preceding crops compared to sole tobacco cultivation. Simultaneously, different preceding crops also affected the expression levels of nitrogen cycling-related genes in tobacco cultivation soil. In the nitrification process, amoA was significantly impacted, with its expression reduced by 64.39% and 72.24% following barley and rapeseed preceding crops, respectively, compared to sole tobacco cultivation. In the denitrification process, except for the narG gene, all other genes were subjected to varying degrees of inhibition when preceded by barley and rapeseed crops. Correlation analysis between soil nitrogen structure and the expression levels of nitrogen cycling-related genes revealed that increased nitrogen levels suppressed the expression of Arch-amoA. Additionally, ammonium nitrogen strongly influenced the expression levels of most soil nitrogen cycling functional genes. In conclusion, preceding crops alter soil nitrogen structure, possibly due to changes in soil microorganisms, and different preceding crops modified the expression levels of nitrogen cycling-related genes in tobacco cultivation soil, consequently affecting the proportions of various nitrogen forms in the soil.

Tobacco, as a member of the Solanaceae family, is typically cultivated in crop rotations with other crops.Different preceding crops, owing to their distinct growth and developmental characteristics, exert various effects on the tobacco soil.Research had shown that certain plants, such as legumes and grasses, possess higher biomass production and root residue, which can significantly enhance soil organic matter content [6][7][8] .Zhou et al. conducted research on four different planting methods, including winter milk vetch, winter rape, winter garlic, and a winter rotation intensification with potato, milk vetch, and rape 9 .They found that the effects of crop rotation on rice yield and soil carbon-nitrogen content varied among different crops.Similarly, Mesfin et al., demonstrated in their study that the rotation of faba bean, field pea, and lentil with wheat influenced wheat's nitrogen uptake efficiency and altered soil nitrogen structure 10 .Preceding crops can also impact soil enzyme activities, particularly those related to nitrogen 11 .Different preceding crops leave behind various types and quantities of root residues in the soil, which influence soil microbial community diversity, richness, and enzyme activity 12 .Study have revealed that certain preceding crops, such as legumes, can increase soil urease and nitrification enzyme activities, facilitating nitrogen conversion and provision 13 .In contrast, other crops may reduce soil enzyme activity, thereby limiting nitrogen supply.Various preceding crops also affect the distribution and forms of nitrogen in the soil 14,15 .Research indicates that some preceding crops can promote the accumulation of nitrogen in the soil, particularly in organic forms such as organic nitrogen and ammonium nitrogen 16 .These nitrogen forms are more favorable for tobacco growth because they not only provide nitrogen nutrition but also reduce competition from nitrate nitrogen for tobacco.Therefore, selecting appropriate preceding crops can improve the distribution of nitrogen forms in the soil, contributing to increased tobacco yield and quality.
Soil nitrogen cycling is an essential component of soil microbial substance cycling and energy transformation, involving a variety of complex and large microorganisms 17 .Among them, nitrogen fixation and nitrification-denitrification processes are crucial components of soil nitrogen cycling.Biological Nitrogen Fixation (BNF) is a specific physiological function of nitrogen-fixing microorganisms, where they reduce atmospheric nitrogen gas (N 2 ) to ammonia nitrogen (NH 3 ) through their nitrogenase enzymes 18 .Research has found that a class of nif genes is responsible for carrying out nitrogen fixation in these microorganisms.nifH is one member of these nitrogen-fixing genes, and its gene sequence is not highly conserved, exhibiting structural variations among different bacterial strains.Therefore, the nifH gene can be employed to study the diversity of nitrogen-fixing bacterial groups in soil, essentially serving as a tool for species identification 19 .On the other hand, nitrification and denitrification processes involve multiple bacteria and soil enzymes.In studies of ammonia oxidation, molecular-level analysis typically targets the amoA gene encoding the AMO subunit.Soil nitrification refers to the process of NH 4 + generating NO 3 − under the action of nitrifying microorganisms in soil, which is actually the oxidation of NH 4 + in Ammonia-Oxidizing Bacteria (AOB) and Ammonia-Oxidizing Archaea (AOA) are oxidized to nitrite NO 2 − and nitrate NO 3 − , which is an important part of measuring the efficiency of the entire nitrogen cycle.It was found that AOB and AOA participated in and led the nitrification process depending on their Ammonia Monooxygenase (AMO).The gene fragment compiling AMO was mainly composed of three subunits, AMO-A, AMO-B and AMO-C.The corresponding genes are amoA, amoB and amoC 20 .Two functional genes, amoA and amoB, are mainly responsible for coding in the soil environment 21 .Among them, amoA gene encoding AMO-A subunit can independently express and synthesize AMO to complete the enzymatic reaction to ensure the smooth nitrification process.Moreover, amoA gene is generally present in AOA and AOB, and has higher conserved and expressed ability than amoB.Therefore, amoA gene is considered to be a typical functional gene in the study of nitrifying microorganisms in soil, and is widely used to analyze the community composition of AOA and AOB 22 .Cultivation experiments conducted by Yu et al., and others have revealed that a relatively low level of dissolved oxygen can promote the transcription of amoA in ammonia-oxidizing bacteria (AOB) 23 .Research by Bollmann et al., and colleagues showed that even after short-term ammonia starvation treatment of Nitrosospira, the mRNA of the amoA gene in Nitrosospira remained present, and ammonia oxidation activity remained at a high level 24 .This indicates that the amoA subunit exhibits both strong adaptability and stability.In the denitrification process, bacteria reduce nitrogen compounds in nitrate (NO 3 − ) to nitrogen gas (N 2 ) through a series of intermediate products (NO 2 − , NO, N 2 O, etc.).Various enzymes, including nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase, participate in this process.Genes such as narG, narZ, and nirK, which encode these enzymes, can reflect the level of nitrification activity in the soil.
We postulate that different preceding crops in Yunnan, China, are likely to induce changes in the nitrogen composition of tobacco-growing soils.We speculate that such alterations may be a contributing factor to modifications in soil nitrogen cycling.It is conceivable that a strong correlation exists between the nitrogen structure of tobacco-growing soils and the nitrogen cycling processes occurring within them.
The field trial employed a randomized block design with three treatments, each with three replicates.Each replicate plot covered an area of 100 m 2 .All preceding crops in the plots were tobacco, with the cultivar being Hongda.The three treatments were as follows: (1) A: No cultivation of any crops; (2) B: Cultivation of barley (cultivar Kunlun 15); (3) C: Cultivation of rapeseed (cultivar Huayou 5).Both Kunlun 15 barley and Huayou 5 rapeseed are widely used local varieties.Barley and rapeseed were sown in December 2022.Row spacing for rapeseed was 25 cm with a plant spacing of 20 cm, while row spacing for barley was 25 cm with a plant spacing of 10 cm.Basal fertilizers were applied before planting, consisting of urea and compound fertilizer.Urea was applied at a rate of 10 kg/acre, and compound fertilizer (15:15:15) was applied at a rate of 12.5 kg/acre.Additional fertilization was carried out in February 2023, with urea applied at a rate of 2.5 kg/acre for both barley and rapeseed.Treatment A involved no crop cultivation and no fertilizer application.All other field management practices followed local field management standards, and harvest was conducted in May 2023.

Soil sample collection and determination
In this experiment, soil sample collection was carried out after the harvest of crops in 2023.For each treatment, ten residual crop remnants were selected after harvesting.The roots were excavated, and adhering soil on the root surfaces was shaken off.A gentle brushing was used to remove and collect the rhizosphere soil still adhering to the roots.A portion of this soil was immediately preserved in liquid nitrogen for the determination of soil nitrogen cycling-related gene expression levels.Another portion was placed in a cool, shaded area for air drying to be used in determining different forms of nitrogen in the soil.
The experiment involved the measurement of four nitrogen forms in the soil, with the method of Liu 25 : total nitrogen (TN), soluble nitrogen (STN), ammonium nitrogen (AMN), and nitrate nitrogen (NIN).In addition, four nitrogen forms were calculated: inorganic nitrogen (IN), organic nitrogen (ON), soluble organic nitrogen (SON), and insoluble organic nitrogen (ION).Total nitrogen in the soil was determined using the continuous flow method (LY/T1228-2015), soluble nitrogen was measured using the alkaline diffusion method (LY/T1228-2015), while ammonium nitrogen and nitrate nitrogen were determined using the continuous flow method (LY/T1228-2015).Inorganic nitrogen = ammonium nitrogen + nitrate nitrogen; organic nitrogen = total nitrogen − inorganic nitrogen; soluble organic nitrogen = soluble nitrogen − inorganic nitrogen, and insoluble organic nitrogen = organic nitrogen − soluble organic nitrogen.

Statistical analysis
Data were statistically analyzed using the SPSS 19.0 software package (IBM, Armonk, USA) for Windows.Differences in all the data were assessed by one-way ANOVA followed by the Tukey's HSD test at P ≤ 0.05.The relationships between genes abundance and soil nitrogen structure were subjected to the Redundancy Discriminant Analyses (RDA).Data were normalized when needed to meet the analysis assumptions.R software (Version 4.0.0) was employed to perform the following analyses.Correlation among the genes abundance and soil nitrogen structure was calculated using principal coordinate analysis (PCoA) in the "vegan" package.The aggregated boosted tree analysis (ABT) using the "gbm" package with 5000 trees for boosting, tenfold crossvalidation, and three-way interactions.

Effects of different former crops on different forms of nitrogen in tobacco-growing soil
The impact of different preceding crops on the nitrogen content in various forms in tobacco-growing soils varies (Fig. 1).However, when data were subjected to dimensionality reduction (Fig. 2), the results showed that A was different from other treatments, while B and C was similarity.Regarding ammonium nitrogen (AMN), there were significant differences among all three treatments, with B > C > A. The AMN content in Treatment B was 82.88% higher than in Treatment A; in Treatment C, it was 63.56% higher than in Treatment A. For nitrate nitrogen (NIN), Treatment A had significantly higher NIN content than Treatments B and C, while there was no significant difference between Treatments B and C. The NIN content in Treatment A was 26.97% higher than in Treatment B and 24.39% higher than in Treatment C. Similarly to NIN, the inorganic nitrogen (IN) content in Treatment A was significantly higher than in Treatments B and C, with no significant difference between Treatments B and C. The IN content in Treatment A was 22.58% higher than in Treatment B and 20.87% higher than in

Effects of different former crops on the expression levels of nitrogen cycling-related genes in tobacco-planting soil
In the soil nitrogen cycling process, nitrification and denitrification are two crucial components.Figure 3 illustrated the expression levels of key genes related to soil nitrogen cycling (nitrification, denitrification, and nitrogen fixation) in tobacco-growing soil with different preceding crops.In the nitrification process, amoA was significantly affected, and the expression of the amoA gene inhibited in Treatments B and C.Under Treatment B, the amoA expression decreased by 64.39% compared to Treatment A, and under Treatment C, the amoA expression decreased by 72.24% compared to Treatment A. Conversely, the expression of Arch-amoA in tobacco-growing soil was minimally affected by the treatments.In the denitrification process, except for the narG gene, all other genes were inhibited to varying degrees in Treatments B and C.Under Treatments B and C, the expression levels of the narG gene in tobacco-growing soil were 42.01 times and 53.73 times higher than in Treatment A, respectively, indicating that Treatments B and C significantly promoted the expression of the narG gene.Meanwhile, in Treatments B and C, the nirK, nirS, norB, and nosZ genes were all inhibited to different extents.

Effects of soil nitrogen structure on expression levels of soil nitrogen cycling-related genes
A correlation analysis was conducted between different forms of nitrogen in tobacco-growing soil and the expression levels of soil nitrogen cycling-related genes (Fig. 4).It was observed that Arch-amoA showed no significant correlations with most forms of nitrogen but exhibited a significant negative correlation with STN.This indicated that an increase in nitrogen content, as an environmental factor, inhibited the expression of Arch-amoA.On the other hand, different forms of nitrogen had a significant impact on the expression of other genes.Particularly, IN and NIN significantly influenced the expression of nitrogen cycling-related genes, except for narG, with which they exhibited a negative correlation.These forms of nitrogen were significantly positively correlated with other genes, including amoA, nirK, nirS, norB, nosZ, and nifH.For amoA, nirK, nirS, norB, nosZ, and nifH, only IN and NIN were positively correlated with gene expression, while other forms of nitrogen exhibited negative correlations or showed no significant relationships.Notably, narG exhibited a positive correlation with ON, SON, and AMN.
The relative influence of the different nitrogen forms on amoA, arch-amoA, narG, nirK, nirS, norB, nosZ and nifH gene copies was determined by the ABT model (Fig. 5), which showed AMN (accounting for 11.3%, 8.7%, 11.7%, 11.8% and 5.9% of the variation) as the major factors determining the abundance of amoA, narG, nirS, norB and nosZ genes, respectively.STN (9.8%) as the major factors determining the abundance of Arch-amoA gene.SON (6.9%) as the major factors determining the abundance of nirK gene.ON (8.7%) as the major factors determining the abundance of nifH gene.

Discussion
Nitrogen forms in tobacco-growing soil were changed by different previous crops Prolonged tobacco cultivation could lead to soil continuous cropping sickness, and crop rotation was the most straightforward and effective method to break these issues.When tobacco was rotated with different crops, the effects on soil improvement vary.Tobacco, as a crop sensitive to continuous cropping, faced significant continuous cropping obstacles in the Yunnan region of China.The results of this study suggested that two planting methods, barley-tobacco and rapeseed-tobacco, significantly increased the content of ammonium nitrogen and soluble organic nitrogen in tobacco-growing soils compared to continuous tobacco planting.Simultaneously, due to the high nitrogen demand of rapeseed and barley during their growth and development stages, the content of nitrate nitrogen and inorganic nitrogen significantly decreased.Therefore, the cultivation of rapeseed and barley as preceding crops altered the proportion of ammonium nitrogen to nitrate nitrogen in tobacco-growing soils.This proportion, in turn, would have a significant impact on the growth and development of subsequent tobacco crops, as suggested by Ghiasi et al., who demonstrated that the ratio of ammonium nitrogen to nitrate nitrogen in tobacco-growing soils affected the metabolic products of tobacco leaves 26 .Furthermore, the study's results indicated that crop rotation patterned with other crops significantly differ in soil nitrogen structure compared to continuous tobacco planting.While the differences in soil nitrogen structure between barley-tobacco and rapeseed-tobacco planting methods were not pronounced, distinctions still existed in specific nitrogen forms.Jiang et al., demonstrated through a 12-year-long-term trial that crop rotation and continuous cropping altered soil microbial diversity, microbial network stability, biomass, nutrient pools, and microbial resources 27 .Menefee et al., found in their study on various grass cropping systems that the soil health score and microbial phospholipid fatty acids (PLFAs) were the lowest in continuous cropping systems 28 .In the context of tobacco research, Wang et al., discovered in their study on mushroom-tobacco rotation that this rotation altered the nutrient structure in soil and also affected the taxonomic groups and dominant species of bacteria and fungi in the soil 29 .Therefore, we concluded that different preceding crops lead to alterations in the nitrogen structure of tobacco-growing soils.Simultaneously, changes in soil microorganisms in tobacco-growing soils might be a contributing factor to the alterations in soil nitrogen structure.

Effects of different preceding crops on the expression of nitrogen cycling-related genes in tobacco-growing soil
Continuous tobacco cultivation not only leaded to nutrient imbalances in tobacco-growing soils but also disrupted soil microbial ecosystems.Soil nitrogen cycling is a critical component of soil microbial substance cycling and energy conversion, involving various complex and extensive microorganisms.This study investigated the nitrogen fixation process (N 2 → NH 3 ), nitrification process (NH 4 + → NO 2 − → NO 3 − ), and denitrification process in tobacco-growing soil under the influence of different preceding crops.The results of this study demonstrated that the choice of different preceding crops leaded to changes in the expression of nitrogen cycling-related genes in tobacco-growing soil.Previous research, such as Iannetta et al., had indicated that crop rotation between legumes and non-legumes could enhance soil nitrogen fixation, while Ma et al., found that wheat and rice rotation could reduce the use of nitrogen fertilizers 30,31 .These studies had collectively shown that crop rotation could improve soil nitrogen fixation capacity.However, in this experiment, the rotation of rapeseed and barley instead decreased the expression of the nifH gene in the soil, thereby www.nature.com/scientificreports/higher in agricultural soils than in non-agricultural soils, and was influenced by soil chemistry under intensive crop rotation but not under monoculture 32 .This process is carried out by ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB).The results of this study showed that rapeseed and barley, as preceding crops, did not significantly alter the expression of arch-amoA in tobacco-growing soils.In contrast, the amoA gene was significantly inhibited, with a more pronounced inhibitory effect observed when rapeseed was used as the preceding crop.Simultaneously, rapeseed and barley as preceding crops reduced nitrous oxide emission by limiting the abundance of nirS and nirK genes.Research by Paungfoo-Lonhienne et al., on legume and sugarcane rotations also indicated that legume crop rotation suppressed the nitrifying microbial community in sugarcanecropping soils 33 .This experiment's findings suggested that different preceding crops altered the expression of nitrogen cycling-related genes in tobacco-growing soil.This change in gene expression, affecting key genes in the nitrification and denitrification processes, subsequently altered the ratio of ammonium nitrogen to nitrate nitrogen in the soil, making it more conducive to tobacco growth.

Impact of soil nitrogen structure on the expression of nitrogen cycling-related genes in soil
Changes in soil nutrient cycling-related genes during soil nutrient cycling processes can be influenced by multiple factors.Research conducted by Lee et al., on forest soils demonstrated that nifH-harboring bacterial communities tended to assemble along environmental gradients 34 .In another study by Hu et al., both gene and transcript abundanced of amoA and nosZ were found to be positively correlated with soil nitrous oxide (N 2 O) emissions 35 .
In our study, we hypothesized that the composition of nitrogen structures in soil, especially in tobacco-growing soils, could influence the expression of nitrogen cycling-related functional genes.The results of this study indicated that Arch-amoA showed no significant correlation with most forms of nitrogen but is significantly negatively correlated with soil total nitrogen.Furthermore, the expression of amoA was also inhibited by most nitrogen forms, suggesting that an increase in nitrogen content acted as an environmental factor that inhibited the conversion of NH 4 + to NO 2 − .Inorganic nitrogen and nitrate nitrogen inhibited the expression of narG, reducing the conversion of NO 3 − to NO 2 − and limiting denitrification.Additionally, inorganic nitrogen and nitrate nitrogen significantly increased the expression of nirK, nirS, norB, and nosZ genes in the soil, promoting the latter stages of denitrification.Research by Mocniak et al., had shown that nitrate nitrogen was not conducive to the synthesis or accumulation of total sugars, reducing sugars, total nitrogen, and chloride ion content in cured tobacco leaves 36 .Therefore, reducing nitrate nitrogen content in tobacco-growing soil could enhance tobacco flavor.This study also found that ammonium nitrogen strongly influenced the expression of most soil nitrogen cycling functional genes.Therefore, when selecting preceding crops for rotation in tobacco-growing soil, it is advisable to choose crops with an appropriate ratio of ammonium nitrogen to nitrate nitrogen in the soil after the preceding crop's harvest.

Conclusion
In this study, barley, rapeseed and non-cultivated crops were used as precursor crops to study the nitrogen structure of tobacco-planting soil and the expression of soil nitrogen cycle related genes.The results showed that different previous crops had different effects on the nitrogen content of different forms in the soil of tobacco cultivation.NH 4 + -N and NO 3 − -N were the two nitrogen forms most affected by the previous crop.The results of this study showed that different previous crops changed the expression levels of nitrogen cycling-related genes in the soil of tobacco cultivation, thus affecting the proportion of various nitrogen forms in the soil.Therefore, when selecting preceding crops for rotation in tobacco cultivation soil, it is advisable to choose crops that result in an appropriate ratio of ammonium nitrogen to nitrate nitrogen in the soil after the preceding crop's harvest.

Figure 4 .Figure 5 .
Figure 4. Effect of soil nitrogen structure on soil gene expression.* Significant at the 0.05 probability level.**Significant at the 0.01 probability level.
The expression of the nirK gene was 48.06% lower in Treatment B and 18.04% lower in Treatment C compared to Treatment A. The nirS gene showed 24.55% lower expression in Treatment B and 49.60% lower expression in Treatment C compared to Treatment A. The norB gene exhibited 50.83% lower expression in Treatment B and 18.04% lower expression in Treatment C compared to Treatment A. The nosZ gene displayed 35.63% lower expression in Treatment B and 48.42% lower expression in Treatment C compared to Treatment A. The expression level of the nifH gene reflected the diversity of nitrogen-fixing bacteria in the soil.In Treatments B and C, the expression of the nifH gene was significantly lower than in Treatment A. Specifically, in Treatment B the nifH gene expression was 23.14% lower than that in Treatment A, and it was nearly absent in Treatment C, with a remarkable 94.27% decrease compared to Treatment A.